Compositions and methods to treat latent viral infections

ABSTRACT

Viral infection is a persistent cause of human disease. Guided nuclease systems target the genomes of viral infections, rendering the viruses incapacitated.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The present application is a continuation of U.S. patent applicationSer. No. 14/725,888, filed May 29, 2015, which claims priority to, andthe benefit of, both U.S. Provisional Patent Application Ser. No.62/005,395, filed May 30, 2014, and U.S. Provisional Patent ApplicationSer. No. 62/029,072, filed Jul. 25, 2014, the contents of which areincorporated by reference.

FIELD OF THE INVENTION

The invention generally relates to compositions and methods forselectively treating viral infections using a guided nuclease system.

BACKGROUND

Viral infections are a significant medical problem. Various antiviraltreatments are available but they generally are directed to interruptingthe replicating cycle of the virus. Thus, a particularly difficultproblem is latent viral infection, as there is no effective treatment toeradicate the virus from host cells. Since latent infection can evadeimmune surveillance and reactivate the lytic cycle at any time, there isa persistent risk throughout the life. The majority of antiviral drugdevelopment has been focused on protein targets and such approaches havenot been successful in eradicating the virus.

One example of a latent viral infection that is a particular problem isthe herpesviridae virus family. Herpes is one of the most widespreadhuman pathogens, with more than 90% of adults having been infected withat least one of the eight subtypes of herpes viruse. Latent infectionpersists in most people; and about 16% of Americans between the ages of14 and 49 are infected with genital herpes, making it one of the mostcommon sexually transmitted diseases. Due to latency, there is no curefor genital herpes or for herpes simplex virus type 2 (HSV-2). Onceinfected, a host carries the herpes virus indefinitely, even when notexpressing symptoms. Similarly, human papillomavirus, or HPV is a commonvirus in the human population, where more than 75% of women and men willhave this type of infection at one point in their life. High-riskoncogenic HPV types are able to integrate into the DNA of the cell thatcan result in cancer, specifically cervical cancer. Similar to theherpesviridae virus family, HPV may remain latent.

The Epstein-Barr virus (EBV), also called human herpesvirus 4 (HHV-4) isanother common virus in humans. Epstein-Barr is known as the cause ofinfectious mononucleosis (glandular fever), and is also associated withparticular forms of cancer, such as Hodgkin's lymphoma, Burkitt'slymphoma, nasopharyngeal carcinoma, and conditions associated with humanimmunodeficiency virus (HIV) such as hairy leukoplakia and centralnervous system lymphomas. There is evidence that infection with thevirus is associated with a higher risk of certain autoimmune diseases,especially dermatomyositis, systemic lupus erythematosus, rheumatoidarthritis, Sjogren's syndrome, and multiple sclerosis. During latency,the EBV genome circularizes and resides in the cell nucleus as episomes.To date, however, no EBV vaccine or treatment exists.

Viruses, such as the herpesviridae virus family, including EBV, and HPVhave the ability to lie dormant within a cell indefinitely and not befully eradicated even after treatment. The result is that the virus canreactivate and begin producing large amounts of viral progeny withoutthe host being infected by any new outside virus. In the latent state,the viral genome persists within the host cells as episomes; stabilizedand floating in the cytoplasm or nucleus. For these latent viruses, ithas not been possible to find therapeutic approaches which completelyeradicate such infections.

SUMMARY

The invention provides methods for selectively treating viral infectionsusing a guided nuclease system. Methods of the invention may be used toremove viral or other foreign genetic material from a host organism,without interfering the integrity of the host's genetic material. Anuclease may be used to target viral nucleic acid, thereby interferingwith viral replication or transcription or even excising the viralgenetic material from the host genome. The nuclease may be specificallytargeted to remove only the viral nucleic acid without acting on hostmaterial either when the viral nucleic acid exists as a particle withinthe cell or when it is integrated into the host genome. Targeting theviral nucleic acid can be done using a sequence-specific moiety such asa guide RNA that targets viral genomic material for destruction by thenuclease and does not target the host cell genome. In some embodiments,a CRISPR/Cas9 nuclease and guide RNA (gRNA) that together target andselectively edit or destroy viral genomic material is used. The CRISPR(clustered regularly interspaced short palindromic repeats) is anaturally-occurring element of the bacterial immune system that protectsbacteria from phage infection. The guide RNA localizes the CRISPR/Cas9complex to a viral target sequence. Binding of the complex localizes theCas9 endonuclease to the viral genomic target sequence causing breaks inthe viral genome. Other nuclease systems can be used including, forexample, zinc finger nucleases, transcription activator-like effectornucleases (TALENs), meganucleases, or any other system that can be usedto degrade or interfere with viral nucleic acid without interfering withthe regular function of the host's genetic material.

In certain aspects, the invention provides a method for treating a viralinfection. The method includes introducing into a cell a nuclease and asequence-specific targeting moiety. The nuclease is targeted to viralnucleic acid by means of the sequence-specific targeting moiety and thenuclease cleaves the viral nucleic acid without interfering with a hostgenome. The nuclease may be, for example, a zinc-finger nuclease, atranscription activator-like effector nuclease, and a meganuclease. In apreferred embodiment, the nuclease is a Cas9 endonuclease and thesequence-specific targeting moiety comprises a guide RNA. The cleavingstep can make one or more single or double stranded breaks in the viralnucleic acid. The method may further include inserting a polynucleotideor re-joining the cleaved ends with a piece of the viral nucleic acidremoved. The host may be a living subject such as a human patient andthe steps may be performed in vivo.

The method may be used to target viral nucleic acid in any form or atany stage in the viral life cycle. For example, the method may be usedto digest viral RNA or DNA. The targeted viral nucleic acid may bepresent in the host cell as independent particles. In a preferredembodiment, the viral infection is latent and the viral nucleic acid isintegrated into the host genome. Any suitable viral nucleic acid may betargeted for cleavage and digestion. In certain embodiments, thetargeted virus includes one or more of herpes simplex virus (HSV)-1,HSV-2, varicella zoster virus (VZV), cytomegalovirus (CMV), humanherpesvirus (HHV)-6, HHV-7, Kaposi's sarcoma-associated herpesvirus(KSHV), JC virus, BK virus, parvovirus b19, adeno-associated virus(AAV), and adenovirus. In some embodiments, the targeted virus orviruses include one or more of Adenovirus, Herpes simplex, type 1,Herpes simplex, type 2, Varicella-zoster virus, Epstein-barr virus,Human cytomegalovirus, Human herpesvirus, type 8, Human papillomavirus,BK virus, JC virus, Smallpox, Hepatitis B virus, Human bocavirus,Parvovirus B19, Human astrovirus, Norwalk virus, coxsackievirus,hepatitis A virus, poliovirus, rhinovirus, Severe acute respiratorysyndrome virus, Hepatitis C virus, yellow fever virus, dengue virus,West Nile virus, Rubella virus, Hepatitis E virus, Humanimmunodeficiency virus (HIV), Influenza virus, Guanarito virus, Juninvirus, Lassa virus, Machupo virus, Sabiá virus, Crimean-Congohemorrhagic fever virus, Ebola virus, Marburg virus, Measles virus,Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Humanmetapneumovirus, Hendra virus, Nipah virus, Rabies virus, Hepatitis D,Rotavirus, Orbivirus, Coltivirus, and Banna virus.

The nuclease and sequence-specific targeting moiety may be introducedinto the cell using a vector. For example, a viral vector that encodesthe nuclease and sequence-specific targeting moiety may be used. Theviral vector may be retrovirus, lentivirus, adenovirus, herpesvirus,poxvirus, alphavirus, vaccinia virus or adeno-associated viruses. Insome embodiments, a non-viral vector is used. A suitable non-viralvector may include, for example, a nanoparticle, a cationic lipid, acationic polymer, metallic nanoparticle, a nanorod, a liposome,microbubbles, a cell-penetrating peptide, a liposphere,polyethyleneglycol (PEG). The cell may be prompted to take up the vectorby, e.g., ultrasound or electroporation.

Aspects of the invention provide a composition for treatment of a viralinfection. The composition includes a nuclease and a sequence-specifictargeting moiety that targets the nuclease to viral nucleic acid in vivowithin a host cell thereby causing the nuclease to cleave the viralnucleic acid without interfering with host nucleic acid. In certainembodiments, the nuclease is a Cas9 endonuclease and thesequence-specific binding module comprises a guide RNA that specificallytargets a portion of a viral genome. The Cas9 endonuclease and the guideRNA may be co-expressed in a host cell infected by a virus. In someembodiments, the nuclease is one selected from the list consisting of azinc-finger nuclease, a transcription activator-like effector nuclease,and a meganuclease.

The viral nucleic acid to be cleaved may include one or more of, e.g.,herpes simplex virus (HSV)-1, HSV-2, varicella zoster virus (VZV),cytomegalovirus (CMV), human herpesvirus (HHV)-6, HHV-7, Kaposi'ssarcoma-associated herpesvirus (KSHV), JC virus, BK virus, parvovirusb19, adeno-associated virus (AAV), and adenovirus, or others.

In some aspects, the invention provides a composition for treatment of aviral infection. The composition includes nucleic acid that encodes anuclease and a sequence-specific targeting moiety that targets thenuclease to viral nucleic acid thereby causing the nuclease to cleavethe viral nucleic acid without interfering with host nucleic acid. Insome embodiments, the sequence-specific targeting moiety uses a guideRNA, which may be complementary to a portion of a viral genome. Theguide RNA may be designed to cause the nuclease to cleave the viralgenome within a feature that is necessary for viral function. Thefeature may be, for example, a viral replication origin, a terminalrepeat, a replication factor binding site, a promoter, a codingsequence, or a repetitive region. The nucleic acid is provided within adelivery vector which may be a viral vector such as an adeno-associatedvirus. The vector could include any of retrovirus, lentivirus,adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia virus, ananoparticle, a cationic lipid, a cationic polymer, a metallicnanoparticle, a nanorod, a liposome, microbubbles, cell-penetratingpeptide, a liposphere, or polyethyleneglycol (PEG).

Methods and compositions of the invention may be used to deliver aCRISPR/gRNA/Cas9 complex to a cell (including entire tissues) that isinfected by a virus. a guide RNA may be designed to target multiplesites on the viral genome in order to disrupt viral nucleic acid andreduce the chance that it will functionally recombine. TheCRISPR/gRNA/Cas9 complexes of the invention can be delivered by viral,non-viral or other methods to effectuate transfection. CRISPR/gRNA/Cas9complexes are preferably designed to target viral genomic material andnot genomic material of the host. In some embodiments, the targetedviral nucleic acid is associated with a virus that causes latentinfection. Latent viruses may be, for example, human immunodeficiencyvirus, human T-cell leukemia virus, Epstein-Barr virus, humancytomegalovirus, human herpesviruses 6 and 7, herpes simplex virus types1 and 2, varicella-zoster virus, measles virus, or human papovaviruses.Aspects of the invention allow for CRISPR/gRNA/Cas9 complexes to bedesigned to target any virus, latent or active.

The presented methods allow for viral genome editing or destruction,which results in the inability of the virus to proliferate and/orinduces apoptosis in infected cells, with no observed cytotoxicity tonon-infected cells. A CRISPR/gRNA/Cas9 complex is designed toselectively target viral genomic material (DNA or RNA), delivering theCRISPR/gRNA/Cas9 complex to a cell containing the viral genome, andcutting the viral genome in order to incapacitate the virus. A viralinfection can thus be treated by targeted disruption of viral genomicfunction or by digestion of viral nucleic acid via one or multiplebreaks caused by targeting sites for endonuclease action in the viralgenome. In some embodiments, methods of the invention may be used fortransfection of a host cell with CRISPR/gRNA/Cas9 to completelysuppressed cell proliferation and/or induce apoptosis in infected cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C represent EBV-targeting CRISPR/Cas9 designs. (FIG. 1A)Scheme of CRISPR/Cas plasmids, adapted from Cong L et al. (2013)Multiplex Genome Engineering Using CRISPR/Cas Systems. Science339:819-823. (FIG. 1B) Effect of oriP on transfection efficiency in Rajicells. Both Cas9 and Cas9-oriP plasmids have a scrambled guide RNA.(FIG. 1C) CRISPR guide RNA targets along the EBV reference genome.Green, red and blue represent three different target sequencecategories.

FIGS. 2A-2F represent CRISPR/Cas9 induced large deletions. (FIG. 2A)Genome context around guide RNA sgEBV2 and PCR primer locations. (FIG.2B) Large deletion induced by sgEBV2. Lane 1-3 are before, 5 days after,and 7 days after sgEBV2 treatment, respectively. (FIG. 2C) Genomecontext around guide RNA sgEBV3/4/5 and PCR primer locations. (FIG. 2D)Large deletions induced by sgEBV3/5 and sgEBV4/5. Lane 1 and 2 are 3F/5RPCR amplicons before and 8 days after sgEBV3/5 treatment. Lane 3 and 4are 4F/5R PCR amplicons before and 8 days after sgEBV4/5 treatment.(FIG. 2E and F) Sanger sequencing confirmed genome cleavage and repairligation 8 days after sgEBV3/5 (FIG. 2E) and sgEBV4/5 (FIG. 2F)treatment. Blue and white background highlights the two ends beforerepair ligation.

FIGS. 3A-3M represent cell proliferation arrest with EBV genomedestruction. (FIG. 3A) Cell proliferation curves after different CRISPRtreatments. Five independent sgEBV 1-7 treatments are shown here. (FIGS.3B-D) Flow cytometry scattering signals before (FIG. 3B), 5 days after(FIG. 3C) and 8 days after (FIG. 3D) sgEBV1-7 treatments. (FIG. 3E-G)Annexin V Alexa647 and DAPI staining results before (FIG. 3E), 5 daysafter (FIG. 3F) and 8 days after (FIG. 3G) sgEBV1-7 treatments. Blue andred correspond to subpopulation P3 and P4 in (FIGS. 3B-D). (FIGS. 3H andI) Microscopy revealed apoptotic cell morphology after sgEBV1-7treatment. (FIGS. 3J-M) Nuclear morphology before (FIG. 3J) and after(FIGS. 3K-M) sgEBV1-7 treatment.

FIGS. 4A-4E represent EBV load quantitation after CRISPR treatment.(FIG. 4A) EBV load after different CRISPR treatments by digital PCR.Cas9 and Cas9-oriP had two replicates, and sgEBV1-7 had 5 replicates.(FIGS. 4B and C) Microscopy of captured single cells for whole-genomeamplification. (FIG. 4D) Histogram of EBV quantitative PCR Ct valuesfrom single cells before treatment. (FIG. 4E) Histogram of EBVquantitative PCR Ct values from single live cells 7 days after sgEBV1-7treatment. Red dash lines in (FIG. 4D) and (FIG. 4E) represent Ct valuesof one EBV genome per cell.

FIG. 5 represents SURVEYOR assay of EBV CRISPR. Lane 1: NEB 100 bpladder; Lane 2: sgEBV1 control; Lane 3: sgEBV1; Lane 4: sgEBV5 control;Lane 5: sgEBV5; Lane 6: sgEBV7 control; Lane 7: sgEBV7; Lane 8: sgEBV4.

FIG. 6 shows CRISPR cytotoxicity test with EBV-negative Burkitt'slymphoma DG-75.

FIG. 7 represents CRISPR cytotoxicity test with primary human lungfibroblast IMR-90.

FIG. 8 shows the use of ZFNs.

FIG. 9 diagrams a method of the invention.

FIG. 10 is a map of an HBV genome.

FIG. 11 shows the results of delivering a viral treatment.

FIG. 12 shows a composition for treating a viral infection.

DETAILED DESCRIPTION

The invention generally relates to compositions and methods forselectively treating viral infections using a guided nuclease system.Methods of the invention are used to incapacitate or disrupt viralnucleic acid within a cell through nuclease activity such as single- ordouble-stranded breaks, cleavage, digestion, or editing. Methods of theinvention may be used for systematically causing large or repeateddeletions in the genome, reducing the probability of reconstructing thefull genome.

i. Treating Infected Cell

FIG. 9 diagrams a method of treating a cell infected with a virus.Methods of the invention are applicable to in vivo treatment of patientsand may be used to remove any viral genetic material such as genes ofvirus associated with a latent viral infection. Methods may be used invitro, e.g., to prepare or treat a cell culture or cell sample. Whenused in vivo, the cell may be any suitable germ line or somatic cell andcompositions of the invention may be delivered to specific parts of apatient's body or be delivered systemically. If delivered systemically,it may be preferable to include within compositions of the inventiontissue-specific promoters. For example, if a patient has a latent viralinfection that is localized to the liver, hepatic tissue-specificpromotors may be included in a plasmid or viral vector that codes for atargeted nuclease.

FIG. 12 shows a composition for treating a viral infection according tocertain embodiments. The composition preferably includes a vector (whichmay be a plasmid, linear DNA, or a viral vector) that codes for anuclease and a targeting moiety (e.g., a gRNA) that targets the nucleaseto viral nucleic acid. The composition may optionally include one ormore of a promoter, replication origin, other elements, or combinationsthereof as described further herein.

ii. Nuclease

Methods of the invention include using a programmable or targetablenuclease to specifically target viral nucleic acid for destruction. Anysuitable targeting nuclease can be used including, for example,zinc-finger nucleases (ZFNs), transcription activator-like effectornucleases (TALENs), clustered regularly interspaced short palindromicrepeat (CRISPR) nucleases, meganucleases, other endo- or exo-nucleases,or combinations thereof. See Schiffer, 2012, Targeted DNA mutagenesisfor the cure of chronic viral infections, J Virol 88(17):8920-8936,incorporated by reference.

CRISPR methodologies employ a nuclease, CRISPR-associated (Cas9), thatcomplexes with small RNAs as guides (gRNAs) to cleave DNA in asequence-specific manner upstream of the protospacer adjacent motif(PAM) in any genomic location. CRISPR may use separate guide RNAs knownas the crRNA and tracrRNA. These two separate RNAs have been combinedinto a single RNA to enable site-specific mammalian genome cuttingthrough the design of a short guide RNA. Cas9 and guide RNA (gRNA) maybe synthesized by known methods. Cas9/guide-RNA (gRNA) uses anon-specific DNA cleavage protein Cas9, and an RNA oligo to hybridize totarget and recruit the Cas9/gRNA complex. See Chang et al., 2013, Genomeediting with RNA-guided Cas9 nuclease in zebrafish embryos, Cell Res23:465-472; Hwang et al., 2013, Efficient genome editing in zebrafishusing a CRISPR-Cas system, Nat. Biotechnol 31:227-229; Xiao et al.,2013, Chromosomal deletions and inversions mediated by TALENS andCRISPR/Cas in zebrafish, Nucl Acids Res 1-11.

CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) isfound in bacteria and is believed to protect the bacteria from phageinfection. It has recently been used as a means to alter gene expressionin eukaryotic DNA, but has not been proposed as an anti-viral therapy ormore broadly as a way to disrupt genomic material. Rather, it has beenused to introduce insertions or deletions as a way of increasing ordecreasing transcription in the DNA of a targeted cell or population ofcells. See for example, Horvath et al., Science (2010) 327:167-170;Terns et al., Current Opinion in Microbiology (2011) 14:321-327; Bhayaet al Annu Rev Genet (2011) 45:273-297; Wiedenheft et al. Nature (2012)482:331-338); Jinek M et al. Science (2012) 337:816-821; Cong L et al.Science (2013) 339:819-823; Jinek M et al. (2013) eLife 2:e00471; Mali Pet al. (2013) Science 339:823-826; Qi LS et al. (2013) Cell152:1173-1183; Gilbert L A et al. (2013) Cell 154:442-451; Yang H et al.(2013) Cell 154:1370-1379; and Wang H et al. (2013) Cell 153:910-918).

In an aspect of the invention, the Cas9 endonuclease causes a doublestrand break in at least two locations in the genome. These two doublestrand breaks cause a fragment of the genome to be deleted. Even ifviral repair pathways anneal the two ends, there will still be adeletion in the genome. One or more deletions using the mechanism willincapacitate the viral genome. The result is that the host cell will befree of viral infection.

In embodiments of the invention, nucleases cleave the genome of thetarget virus. A nuclease is an enzyme capable of cleaving thephosphodiester bonds between the nucleotide subunits of nucleic acids.Endonucleases are enzymes that cleave the phosphodiester bond within apolynucleotide chain. Some, such as Deoxyribonuclease I, cut DNArelatively nonspecifically (without regard to sequence), while many,typically called restriction endonucleases or restriction enzymes,cleave only at very specific nucleotide sequences. In a preferredembodiment of the invention, the Cas9 nuclease is incorporated into thecompositions and methods of the invention, however, it should beappreciated that any nuclease may be utilized.

In preferred embodiments of the invention, the Cas9 nuclease is used tocleave the genome. The Cas9 nuclease is capable of creating a doublestrand break in the genome. The Cas9 nuclease has two functionaldomains: RuvC and HNH, each cutting a different strand. When both ofthese domains are active, the Cas9 causes double strand breaks in thegenome.

In some embodiments of the invention, insertions into the genome can bedesigned to cause incapacitation, or altered genomic expression.Additionally, insertions/deletions are also used to introduce apremature stop codon either by creating one at the double strand breakor by shifting the reading frame to create one downstream of the doublestrand break. Any of these outcomes of the NHEJ repair pathway can beleveraged to disrupt the target gene. The changes introduced by the useof the CRISPR/gRNA/Cas9 system are permanent to the genome.

In some embodiments of the invention, at least one insertion is causedby the CRISPR/gRNA/Cas9 complex. In a preferred embodiment, numerousinsertions are caused in the genome, thereby incapacitating the virus.In an aspect of the invention, the number of insertions lowers theprobability that the genome may be repaired.

In some embodiments of the invention, at least one deletion is caused bythe CRISPR/gRNA/Cas9 complex. In a preferred embodiment, numerousdeletions are caused in the genome, thereby incapacitating the virus. Inan aspect of the invention, the number of deletions lowers theprobability that the genome may be repaired. In a highly-preferredembodiment, the CRISPR/Cas9/gRNA system of the invention causessignificant genomic disruption, resulting in effective destruction ofthe viral genome, while leaving the host genome intact.

TALENs uses a nonspecific DNA-cleaving nuclease fused to a DNA-bindingdomain that can be to target essentially any sequence. For TALENtechnology, target sites are identified and expression vectors are made.Linearized expression vectors (e.g., by Notl) may be used as templatefor mRNA synthesis. A commercially available kit may be use such as themMESSAGE mMACHINE SP6 transcription kit from Life Technologies(Carlsbad, Calif.). See Joung & Sander, 2013, TALENs: a widelyapplicable technology for targeted genome editing, Nat Rev Mol Cell Bio14:49-55.

TALENs and CRISPR methods provide one-to-one relationship to the targetsites, i.e. one unit of the tandem repeat in the TALE domain recognizesone nucleotide in the target site, and the crRNA, gRNA, or sgRNA ofCRISPR/Cas system hybridizes to the complementary sequence in the DNAtarget. Methods can include using a pair of TALENs or a Cas9 proteinwith one gRNA to generate double-strand breaks in the target. The breaksare then repaired via non-homologous end joining or homologousrecombination (HR).

FIG. 8 shows ZFN being used to cut viral nucleic acid. Briefly, the ZFNmethod includes introducing into the infected host cell at least onevector (e.g., RNA molecule) encoding a targeted ZFN 305 and, optionally,at least one accessory polynucleotide. See, e.g., U.S. Pub. 2011/0023144to Weinstein, incorporated by reference The cell includes targetsequence 311. The cell is incubated to allow expression of the ZFN 305,wherein a double-stranded break 317 is introduced into the targetedchromosomal sequence 311 by the ZFN 305. In some embodiments, a donorpolynucleotide or exchange polynucleotide 321 is introduced. Swapping aportion of the viral nucleic acid with irrelevant sequence can fullyinterfere transcription or replication of the viral nucleic acid. TargetDNA 311 along with exchange polynucleotide 321 may be repaired by anerror-prone non-homologous end joining DNA repair process or ahomology-directed DNA repair process.

Typically, a ZFN comprises a DNA binding domain (i.e., zinc finger) anda cleavage domain (i.e., nuclease) and this gene may be introduced asmRNA (e.g., 5′ capped, polyadenylated, or both). Zinc finger bindingdomains may be engineered to recognize and bind to any nucleic acidsequence of choice. See, e.g., Qu et al., 2013, Zinc-finger-nucleasesmediate specific and efficient excision of HIV-1 proviral DAN frominfected and latently infected human T cells, Nucl Ac Res41(16):7771-7782, incorporated by reference. An engineered zinc fingerbinding domain may have a novel binding specificity compared to anaturally-occurring zinc finger protein. Engineering methods include,but are not limited to, rational design and various types of selection.A zinc finger binding domain may be designed to recognize a target DNAsequence via zinc finger recognition regions (i.e., zinc fingers). Seefor example, U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242,incorporated by reference. Exemplary methods of selecting a zinc fingerrecognition region may include phage display and two-hybrid systems, andare disclosed in U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S.Pat. No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,410,248;U.S. Pat. No. 6,140,466; U.S. Pat. No. 6,200,759; and U.S. Pat. No.6,242,568, each of which is incorporated by reference.

A ZFN also includes a cleavage domain. The cleavage domain portion ofthe ZFNs may be obtained from any suitable endonuclease or exonucleasesuch as restriction endonucleases and homing endonucleases. See, forexample, Belfort & Roberts, 1997, Homing endonucleases: keeping thehouse in order, Nucleic Acids Res 25(17):3379-3388. A cleavage domainmay be derived from an enzyme that requires dimerization for cleavageactivity. Two ZFNs may be required for cleavage, as each nucleasecomprises a monomer of the active enzyme dimer. Alternatively, a singleZFN may comprise both monomers to create an active enzyme dimer.Restriction endonucleases present may be capable of sequence-specificbinding and cleavage of DNA at or near the site of binding. Certainrestriction enzymes (e.g., Type IIS) cleave DNA at sites removed fromthe recognition site and have separable binding and cleavage domains.For example, the Type IIS enzyme FokI, active as a dimer, catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. The FokI enzyme used in a ZFN may be considered a cleavagemonomer. Thus, for targeted double-stranded cleavage using a FokIcleavage domain, two ZFNs, each comprising a FokI cleavage monomer, maybe used to reconstitute an active enzyme dimer. See Wah, et al., 1998,Structure of FokI has implications for DNA cleavage, PNAS95:10564-10569; U.S. Pat. No. 5,356,802; U.S. Pat. No. 5,436,150; U.S.Pat. No. 5,487,994; U.S. Pub. 2005/0064474; U.S. Pub. 2006/0188987; andU.S. Pub. 2008/0131962, each incorporated by reference.

Virus targeting using ZFN may include introducing at least one donorpolynucleotide comprising a sequence into the cell. A donorpolynucleotide preferably includes the sequence to be introduced flankedby an upstream and downstream sequence that share sequence similaritywith either side of the site of integration in the chromosome. Theupstream and downstream sequences in the donor polynucleotide areselected to promote recombination between the chromosomal sequence ofinterest and the donor polynucleotide. Typically, the donorpolynucleotide will be DNA. The donor polynucleotide may be a DNAplasmid, a bacterial artificial chromosome (BAC), a yeast artificialchromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment,a naked nucleic acid, and may employ a delivery vehicle such as aliposome. The sequence of the donor polynucleotide may include exons,introns, regulatory sequences, or combinations thereof. The doublestranded break is repaired via homologous recombination with the donorpolynucleotide such that the desired sequence is integrated into thechromosome. In the ZFN-mediated process, a double stranded breakintroduced into the target sequence by the ZFN is repaired, viahomologous recombination with the exchange polynucleotide, such that thesequence in the exchange polynucleotide may be exchanged with a portionof the target sequence. The presence of the double stranded breakfacilitates homologous recombination and repair of the break. Theexchange polynucleotide may be physically integrated or, alternatively,the exchange polynucleotide may be used as a template for repair of thebreak, resulting in the exchange of the sequence information in theexchange polynucleotide with the sequence information in that portion ofthe target sequence. Thus, a portion of the viral nucleic acid may beconverted to the sequence of the exchange polynucleotide. ZFN methodscan include using a vector to deliver a nucleic acid molecule encoding aZFN and, optionally, at least one exchange polynucleotide or at leastone donor polynucleotide to the infected cell.

Meganucleases are endodeoxyribonucleases characterized by a largerecognition site (double-stranded DNA sequences of 12 to 40 base pairs);as a result this site generally occurs only once in any given genome.For example, the 18-base pair sequence recognized by the I-SceImeganuclease would on average require a genome twenty times the size ofthe human genome to be found once by chance (although sequences with asingle mismatch occur about three times per human-sized genome).Meganucleases are therefore considered to be the most specific naturallyoccurring restriction enzymes. Meganucleases can be divided into fivefamilies based on sequence and structure motifs: LAGLIDADG, GIY-YIG,HNH, His-Cys box and PD-(D/E)XK. The most well studied family is that ofthe LAGLIDADG proteins, which have been found in all kingdoms of life,generally encoded within introns or inteins although freestandingmembers also exist. The sequence motif, LAGLIDADG, represents anessential element for enzymatic activity. Some proteins contained onlyone such motif, while others contained two; in both cases the motifswere followed by ˜75-200 amino acid residues having little to nosequence similarity with other family members. Crystal structuresillustrates mode of sequence specificity and cleavage mechanism for theLAGLIDADG family: (i) specificity contacts arise from the burial ofextended β-strands into the major groove of the DNA, with the DNAbinding saddle having a pitch and contour mimicking the helical twist ofthe DNA; (ii) full hydrogen bonding potential between the protein andDNA is never fully realized; (iii) cleavage to generate thecharacteristic 4-nt 3′-OH overhangs occurs across the minor groove,wherein the scissile phosphate bonds are brought closer to the proteincatalytic core by a distortion of the DNA in the central “4-base”region; (iv) cleavage occurs via a proposed two-metal mechanism,sometimes involving a unique “metal sharing” paradigm; (v) and finally,additional affinity and/or specificity contacts can arise from “adapted”scaffolds, in regions outside the core α/β fold. See Silva et al., 2011,Meganucleases and other tools for targeted genome engineering, Curr GeneTher 11(1):11-27, incorporated by reference.

In some embodiments of the invention, a template sequence is insertedinto the genome. In order to introduce nucleotide modifications togenomic DNA, a DNA repair template containing the desired sequence mustbe present during homology directed repair (HDR). The DNA template isnormally transfected into the cell along with the gRNA/Cas9. The lengthand binding position of each homology arm is dependent on the size ofthe change being introduced. In the presence of a suitable template, HDRcan introduce significant changes at the Cas9 induced double strandbreak.

Some embodiments of the invention may utilize modified version of anuclease. Modified versions of the Cas9 enzyme containing a singleinactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’.With only one active nuclease domain, the Cas9 nickase cuts only onestrand of the target DNA, creating a single-strand break or ‘nick’.Similar to the inactive dCas9 (RuvC- and HNH-), a Cas9 nickase is stillable to bind DNA based on gRNA specificity, though nickases will onlycut one of the DNA strands. The majority of CRISPR plasmids are derivedfrom S. pyogenes and the RuvC domain can be inactivated by a D10Amutation and the HNH domain can be inactivated by an H840A mutation.

A single-strand break, or nick, is normally quickly repaired through theHDR pathway, using the intact complementary DNA strand as the template.However, two proximal, opposite strand nicks introduced by a Cas9nickase are treated as a double strand break, in what is often referredto as a ‘double nick’ or ‘dual nickase’ CRISPR system. A double-nickinduced double strain break can be repaired by either NHEJ or HDRdepending on the desired effect on the gene target. At these doublestrain breaks, insertions and deletions are caused by the CRISPR/Cas9complex. In an aspect of the invention, a deletion is caused bypositioning two double strand breaks proximate to one another, therebycausing a fragment of the genome to be deleted.

iii. Targeting Moiety

A nuclease may use the targeting specificity of a guide RNA (gRNA). Asdiscussed below, guide RNAs or single guide RNAs are specificallydesigned to target a virus genome.

A CRISPR/Cas9 gene editing complex of the invention works optimally witha guide RNA that targets the viral genome. Guide RNA (gRNA) (whichincludes single guide RNA (sgRNA), crisprRNA (crRNA), transactivatingRNA (tracrRNA), any other targeting oligo, or any combination thereof)leads the CRISPR/Cas9 complex to the viral genome in order to causeviral genomic disruption. In an aspect of the invention,CRISPR/Cas9/gRNA complexes are designed to target specific viruseswithin a cell. It should be appreciated that any virus can be targetedusing the composition of the invention. Identification of specificregions of the virus genome aids in development and designing ofCRISPR/Cas9/gRNA complexes.

In an aspect of the invention, the CRISPR/Cas9/gRNA complexes aredesigned to target latent viruses within a cell. Once transfected withina cell, the CRISPR/Cas9/gRNA complexes cause repeated insertions ordeletions to render the genome incapacitated, or due to number ofinsertions or deletions, the probability of repair is significantlyreduced.

As an example, the Epstein-Barr virus (EBV), also called humanherpesvirus 4 (HHV-4) is inactivated in cells by a CRISPR/Cas9/gRNAcomplex of the invention. EBV is a virus of the herpes family, and isone of the most common viruses in humans. The virus is approximately 122nm to 180 nm in diameter and is composed of a double helix of DNAwrapped in a protein capsid. In this example, the Raji cell line servesas an appropriate in vitro model. The Raji cell line is the firstcontinuous human cell line from hematopoietic origin and cell linesproduce an unusual strain of Epstein-Barr virus while being one of themost extensively studied EBV models. To target the EBV genomes in theRaji cells, a CRISPR/Cas9 complex with specificity for EBV is needed.The design of EBV-targeting CRISPR/Cas9 plasmids consisting of a U6promoter driven chimeric guide RNA (sgRNA) and a ubiquitous promoterdriven Cas9 that were obtained from Addgene, Inc. Commercially availableguide RNAs and Cas9 nucleases may be used with the present invention. AnEGFP marker fused after the Cas9 protein allowed selection ofCas9-positive cells (FIG. 1A).

In an aspect of the invention, guide RNAs are designed, whether or notcommercially purchased, to target a specific viral genome. The viralgenome is identified and guide RNA to target selected portions of theviral genome are developed and incorporated into the composition of theinvention. In an aspect of the invention, a reference genome of aparticular strain of the virus is selected for guide RNA design.

For example, guide RNAs that target the EBV genome are a component ofthe system in the present example. In relation to EBV, for example, thereference genome from strain B95-8 was used as a design guide. Within agenome of interest, such as EBV, selected regions, or genes aretargeted. For example, six regions can be targeted with seven guide RNAdesigns for different genome editing purposes (FIG. 1C and Table S1). Inrelation to EBV, EBNA1 is the only nuclear Epstein-Barr virus (EBV)protein expressed in both latent and lytic modes of infection. WhileEBNA1 is known to play several important roles in latent infection,EBNA1 is crucial for many EBV functions including gene regulation andlatent genome replication. Therefore, guide RNAs sgEBV4 and sgEBV5 wereselected to target both ends of the EBNA1 coding region in order toexcise this whole region of the genome. These “structural” targetsenable systematic digestion of the EBV genome into smaller pieces.EBNA3C and LMP1 are essential for host cell transformation, and guideRNAs sgEBV3 and sgEBV7 were designed to target the 5′ exons of these twoproteins respectively.

iv. Introduce to Cell

Methods of the invention include introducing into a cell a nuclease anda sequence-specific targeting moiety. The nuclease is targeted to viralnucleic acid by means of the sequence-specific targeting moiety where itthen cleaves the viral nucleic acid without interfering with a hostgenome. Any suitable method can be used to deliver the nuclease to theinfected cell or tissue. For example, the nuclease or the gene encodingthe nuclease may be delivered by injection, orally, or by hydrodynamicdelivery. The nuclease or the gene encoding the nuclease may bedelivered to systematic circulation or may be delivered or otherwiselocalized to a specific tissue type. The nuclease or gene encoding thenuclease may be modified or programmed to be active under only certainconditions such as by using a tissue-specific promoter so that theencoded nuclease is preferentially or only transcribed in certain tissuetypes.

In some embodiments, specific CRISPR/Cas9/gRNA complexes are introducedinto a cell. A guide RNA is designed to target at least one category ofsequences of the viral genome. In addition to latent infections thisinvention can also be used to control actively replicating viruses bytargeting the viral genome before it is packaged or after it is ejected.

In some embodiments, a cocktail of guide RNAs may be introduced into acell. The guide RNAs are designed to target numerous categories ofsequences of the viral genome. By targeting several areas along thegenome, the double strand break at multiple locations fragments thegenome, lowering the possibility of repair. Even with repair mechanisms,the large deletions render the virus incapacitated.

In some embodiments, several guide RNAs are added to create a cocktailto target different categories of sequences. For example, two, five,seven or eleven guide RNAs may be present in a CRISPR cocktail targetingthree different categories of sequences. However, any number of gRNAsmay be introduced into a cocktail to target categories of sequences. Inpreferred embodiments, the categories of sequences are important forgenome structure, host cell transformation, and infection latency,respectively.

In some aspects of the invention, in vitro experiments allow for thedetermination of the most essential targets within a viral genome. Forexample, to understand the most essential targets for effectiveincapacitation of a genome, subsets of guide RNAs are transfected intomodel cells. Assays can determine which guide RNAs or which cocktail isthe most effective at targeting essential categories of sequences.

For example, in the case of the EBV genome targeting, seven guide RNAsin the CRISPR cocktail targeted three different categories of sequenceswhich are identified as being important for EBV genome structure, hostcell transformation, and infection latency, respectively. To understandthe most essential targets for effective EBV treatment, Raji cells weretransfected with subsets of guide RNAs. Although sgEBV4/5 reduced theEBV genome by 85%, they could not suppress cell proliferation aseffectively as the full cocktail (FIG. 3A). Guide RNAs targeting thestructural sequences (sgEBV1/2/6) could stop cell proliferationcompletely, despite not eliminating the full EBV load (26% decrease).Given the high efficiency of genome editing and the proliferation arrest(FIG. 2), it was suspect that the residual EBV genome signature insgEBV1/2/6 was not due to intact genomes but to free-floating DNA thathas been digested out of the EBV genome, i.e. as a false positive.

Once CRISPR/Cas9/gRNA complexes are constructed, the complexes areintroduced into a cell. It should be appreciated that complexes can beintroduced into cells in an in vitro model or an in vivo model. In anaspect of the invention, CRISPR/Cas9/gRNA complexes are designed to notleave intact genomes of a virus after transfection and complexes aredesigned for efficient transfection.

Aspects of the invention allow for CRISPR/Cas9/gRNA to be transfectedinto cells by various methods, including viral vectors and non-viralvectors. Viral vectors may include retroviruses, lentiviruses,adenoviruses, and adeno-associated viruses. It should be appreciatedthat any viral vector may be incorporated into the present invention toeffectuate delivery of the CRISPR/Cas9/gRNA complex into a cell. Someviral vectors may be more effective than others, depending on theCRISPR/Cas9/gRNA complex designed for digestion or incapacitation. In anaspect of the invention, the vectors contain essential components suchas origin of replication, which is necessary for the replication andmaintenance of the vector in the host cell.

In an aspect of the invention, viral vectors are used as deliveryvectors to deliver the complexes into a cell. Use of viral vectors asdelivery vectors are known in the art. See for example U.S. Pub.2009/0017543 to Wilkes et al., the contents of which are incorporated byreference.

A retrovirus is a single-stranded RNA virus that stores its nucleic acidin the form of an mRNA genome (including the 5′ cap and 3′ PolyA tail)and targets a host cell as an obligate parasite. In some methods in theart, retroviruses have been used to introduce nucleic acids into a cell.Once inside the host cell cytoplasm the virus uses its own reversetranscriptase enzyme to produce DNA from its RNA genome, the reverse ofthe usual pattern, thus retro (backwards). This new DNA is thenincorporated into the host cell genome by an integrase enzyme, at whichpoint the retroviral DNA is referred to as a provirus. For example, therecombinant retroviruses such as the Moloney murine leukemia virus havethe ability to integrate into the host genome in a stable fashion. Theycontain a reverse transcriptase that allows integration into the hostgenome. Retroviral vectors can either be replication-competent orreplication-defective. In some embodiments of the invention,retroviruses are incorporated to effectuate transfection into a cell,however the CRISPR/Cas9/gRNA complexes are designed to target the viralgenome.

In some embodiments of the invention, lentiviruses, which are a subclassof retroviruses, are used as viral vectors. Lentiviruses can be adaptedas delivery vehicles (vectors) given their ability to integrate into thegenome of non-dividing cells, which is the unique feature oflentiviruses as other retroviruses can infect only dividing cells. Theviral genome in the form of RNA is reverse-transcribed when the virusenters the cell to produce DNA, which is then inserted into the genomeat a random position by the viral integrase enzyme. The vector, nowcalled a provirus, remains in the genome and is passed on to the progenyof the cell when it divides.

As opposed to lentiviruses, adenoviral DNA does not integrate into thegenome and is not replicated during cell division. Adenovirus and therelated AAV would be potential approaches as delivery vectors since theydo not integrate into the host's genome. In some aspects of theinvention, only the viral genome to be targeted is effected by theCRISPR/Cas9/gRNA complexes, and not the host's cells. Adeno-associatedvirus (AAV) is a small virus that infects humans and some other primatespecies. AAV can infect both dividing and non-dividing cells and mayincorporate its genome into that of the host cell. For example, becauseof its potential use as a gene therapy vector, researchers have createdan altered AAV called self-complementary adeno-associated virus (scAAV).Whereas AAV packages a single strand of DNA and requires the process ofsecond-strand synthesis, scAAV packages both strands which annealtogether to form double stranded DNA. By skipping second strandsynthesis scAAV allows for rapid expression in the cell. Otherwise,scAAV carries many characteristics of its AAV counterpart. Methods ofthe invention may incorporate herpesvirus, poxvirus, alphavirus, orvaccinia virus as a means of delivery vectors.

In certain embodiments of the invention, non-viral vectors may be usedto effectuate transfection. Methods of non-viral delivery of nucleicacids include lipofection, nucleofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386,4,946,787; and 4,897,355) and lipofection reagents are sold commercially(e.g., Transfectam and Lipofectin). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those described in U.S. Pat. No. 7,166,298 toJessee or U.S. Pat. No. 6,890,554 to Jesse, the contents of each ofwhich are incorporated by reference. Delivery can be to cells (e.g. invitro or ex vivo administration) or target tissues (e.g. in vivoadministration).

Synthetic vectors are typically based on cationic lipids or polymerswhich can complex with negatively charged nucleic acids to formparticles with a diameter in the order of 100 nm. The complex protectsnucleic acid from degradation by nuclease. Moreover, cellular and localdelivery strategies have to deal with the need for internalization,release, and distribution in the proper subcellular compartment.Systemic delivery strategies encounter additional hurdles, for example,strong interaction of cationic delivery vehicles with blood components,uptake by the reticuloendothelial system, kidney filtration, toxicityand targeting ability of the carriers to the cells of interest.Modifying the surfaces of the cationic non-virals can minimize theirinteraction with blood components, reduce reticuloendothelial systemuptake, decrease their toxicity and increase their binding affinity withthe target cells. Binding of plasma proteins (also termed opsonization)is the primary mechanism for RES to recognize the circulatingnanoparticles. For example, macrophages, such as the Kupffer cells inthe liver, recognize the opsonized nanoparticles via the scavengerreceptor.

In some embodiments of the invention, non-viral vectors are modified toeffectuate targeted delivery and transfection. PEGylation (i.e.modifying the surface with polyethyleneglycol) is the predominant methodused to reduce the opsonization and aggregation of non-viral vectors andminimize the clearance by reticuloendothelial system, leading to aprolonged circulation lifetime after intravenous (i.v.) administration.PEGylated nanoparticles are therefore often referred as “stealth”nanoparticles. The nanoparticles that are not rapidly cleared from thecirculation will have a chance to encounter infected cells.

However, PEG on the surface can decrease the uptake by target cells andreduce the biological activity. Therefore, to attach targeting ligand tothe distal end of the PEGylated component is necessary; the ligand isprojected beyond the PEG “shield” to allow binding to receptors on thetarget cell surface. When cationic liposome is used as gene carrier, theapplication of neutral helper lipid is helpful for the release ofnucleic acid, besides promoting hexagonal phase formation to enableendosomal escape. In some embodiments of the invention, neutral oranionic liposomes are developed for systemic delivery of nucleic acidsand obtaining therapeutic effect in experimental animal model. Designingand synthesizing novel cationic lipids and polymers, and covalently ornoncovalently binding gene with peptides, targeting ligands, polymers,or environmentally sensitive moieties also attract many attentions forresolving the problems encountered by non-viral vectors. The applicationof inorganic nanoparticles (for example, metallic nanoparticles, ironoxide, calcium phosphate, magnesium phosphate, manganese phosphate,double hydroxides, carbon nanotubes, and quantum dots) in deliveryvectors can be prepared and surface-functionalized in many differentways.

In some embodiments of the invention, targeted controlled-releasesystems responding to the unique environments of tissues and externalstimuli are utilized. Gold nanorods have strong absorption bands in thenear-infrared region, and the absorbed light energy is then convertedinto heat by gold nanorods, the so-called ‘photothermal effect’. Becausethe near-infrared light can penetrate deeply into tissues, the surfaceof gold nanorod could be modified with nucleic acids for controlledrelease. When the modified gold nanorods are irradiated by near-infraredlight, nucleic acids are released due to thermo-denaturation induced bythe photothermal effect. The amount of nucleic acids released isdependent upon the power and exposure time of light irradiation.

In some embodiments of the invention, liposomes are used to effectuatetransfection into a cell or tissue. The pharmacology of a liposomalformulation of nucleic acid is largely determined by the extent to whichthe nucleic acid is encapsulated inside the liposome bilayer.Encapsulated nucleic acid is protected from nuclease degradation, whilethose merely associated with the surface of the liposome is notprotected. Encapsulated nucleic acid shares the extended circulationlifetime and biodistribution of the intact liposome, while those thatare surface associated adopt the pharmacology of naked nucleic acid oncethey disassociate from the liposome.

In some embodiments, the complexes of the invention are encapsulated ina liposome. Unlike small molecule drugs, nucleic acids cannot crossintact lipid bilayers, predominantly due to the large size andhydrophilic nature of the nucleic acid. Therefore, nucleic acids may beentrapped within liposomes with conventional passive loadingtechnologies, such as ethanol drop method (as in SALP), reverse-phaseevaporation method, and ethanol dilution method (as in SNALP).

In some embodiments, linear polyethylenimine (L-PEI) is used as anon-viral vector due to its versatility and comparatively hightransfection efficiency. L-PEI has been used to efficiently delivergenes in vivo into a wide range of organs such as lung, brain, pancreas,retina, bladder as well as tumor. L-PEI is able to efficiently condense,stabilize and deliver nucleic acids in vitro and in vivo.

Low-intensity ultrasound in combination with microbubbles has recentlyacquired much attention as a safe method of gene delivery. Ultrasoundshows tissue-permeabilizing effect. It is non-invasive andsite-specific, and could make it possible to destroy tumor cells aftersystemic delivery, while leave nontargeted organs unaffected.Ultrasound-mediated microbubbles destruction has been proposed as aninnovative method for noninvasive delivering of drugs and nucleic acidsto different tissues. Microbubbles are used to carry a drug or geneuntil a specific area of interest is reached, and then ultrasound isused to burst the microbubbles, causing site-specific delivery of thebioactive materials. Furthermore, the ability of albumin-coatedmicrobubbles to adhere to vascular regions with glycocalix damage orendothelial dysfunction is another possible mechanism to deliver drugseven in the absence of ultrasound. See Tsutsui et al., 2004, The use ofmicrobubbles to target drug delivery, Cardiovasc Ultrasound 2:23, thecontents of which are incorporated by reference. In ultrasound-triggereddrug delivery, tissue-permeabilizing effect can be potentiated usingultrasound contrast agents, gas-filled microbubbles. The use ofmicrobubbles for delivery of nucleic acids is based on the hypothesisthat destruction of DNA-loaded microbubbles by a focused ultrasound beamduring their microvascular transit through the target area will resultin localized transduction upon disruption of the microbubble shell whilesparing non-targeted areas.

Besides ultrasound-mediated delivery, magnetic targeting delivery couldbe used for delivery. Magnetic nanoparticles are usually entrapped ingene vectors for imaging the delivery of nucleic acid. Nucleic acidcarriers can be responsive to both ultrasound and magnetic fields, i.e.,magnetic and acoustically active lipospheres (MAALs). The basic premiseis that therapeutic agents are attached to, or encapsulated within, amagnetic micro- or nanoparticle. These particles may have magnetic coreswith a polymer or metal coating which can be functionalized, or mayconsist of porous polymers that contain magnetic nanoparticlesprecipitated within the pores. By functionalizing the polymer or metalcoating it is possible to attach, for example, cytotoxic drugs fortargeted chemotherapy or therapeutic DNA to correct a genetic defect.Once attached, the particle/therapeutic agent complex is injected intothe bloodstream, often using a catheter to position the injection sitenear the target. Magnetic fields, generally from high-field,high-gradient, rare earth magnets are focused over the target site andthe forces on the particles as they enter the field allow them to becaptured and extravasated at the target.

Synthetic cationic polymer-based nanoparticles (˜100 nm diameter) havebeen developed that offer enhanced transfection efficiency combined withreduced cytotoxicity, as compared to traditional liposomes. Theincorporation of distinct layers composed of lipid molecules withvarying physical and chemical characteristics into the polymernanoparticle formulation resulted in improved efficiency through betterfusion with cell membrane and entry into the cell, enhanced release ofmolecules inside the cell, and reduced intracellular degradation ofnanoparticle complexes.

In some embodiments, the complexes are conjugated to nano-systems forsystemic therapy, such as liposomes, albumin-based particles, PEGylatedproteins, biodegradable polymer-drug composites, polymeric micelles,dendrimers, among others. See Davis et al., 2008, Nanotherapeuticparticles: an emerging treatment modality for cancer, Nat Rev DrugDiscov. 7(9):771-782, incorporated by reference. Long circulatingmacromolecular carriers such as liposomes, can exploit the enhancedpermeability and retention effect for preferential extravasation fromtumor vessels. In certain embodiments, the complexes of the inventionare conjugated to or encapsulated into a liposome or polymerosome fordelivery to a cell. For example, liposomal anthracyclines have achievedhighly efficient encapsulation, and include versions with greatlyprolonged circulation such as liposomal daunorubicin and pegylatedliposomal doxorubicin. See Krishna et al., Carboxymethylcellulose-sodiumbased transdermal drug delivery system for propranolol, J PharmPharmacol. 1996 April; 48(4):367-70.

Liposomal delivery systems provide stable formulation, provide improvedpharmacokinetics, and a degree of ‘passive’ or ‘physiological’ targetingto tissues. Encapsulation of hydrophilic and hydrophobic materials, suchas potential chemotherapy agents, are known. See for example U.S. Pat.No. 5,466,468 to Schneider, which discloses parenterally administrableliposome formulation comprising synthetic lipids; U.S. Pat. No.5,580,571, to Hostetler et al. which discloses nucleoside analoguesconjugated to phospholipids; U.S. Pat. No. 5,626,869 to Nyqvist, whichdiscloses pharmaceutical compositions wherein the pharmaceuticallyactive compound is heparin or a fragment thereof contained in a definedlipid system comprising at least one amphiphatic and polar lipidcomponent and at least one nonpolar lipid component.

Liposomes and polymerosomes can contain a plurality of solutions andcompounds. In certain embodiments, the complexes of the invention arecoupled to or encapsulated in polymersomes. As a class of artificialvesicles, polymersomes are tiny hollow spheres that enclose a solution,made using amphiphilic synthetic block copolymers to form the vesiclemembrane. Common polymersomes contain an aqueous solution in their coreand are useful for encapsulating and protecting sensitive molecules,such as drugs, enzymes, other proteins and peptides, and DNA and RNAfragments. The polymersome membrane provides a physical barrier thatisolates the encapsulated material from external materials, such asthose found in biological systems. Polymerosomes can be generated fromdouble emulsions by known techniques, see Lorenceau et al., 2005,Generation of Polymerosomes from Double-Emulsions, Langmuir21(20):9183-6, incorporated by reference.

Some embodiments of the invention provide for a gene gun or a biolisticparticle delivery system. A gene gun is a device for injecting cellswith genetic information, where the payload may be an elemental particleof a heavy metal coated with plasmid DNA. This technique may also bereferred to as bioballistics or biolistics. Gene guns have also beenused to deliver DNA vaccines. The gene gun is able to transfect cellswith a wide variety of organic and non-organic species, such as DNAplasmids, fluorescent proteins, dyes, etc.

Aspects of the invention provide for numerous uses of delivery vectors.Selection of the delivery vector is based upon the cell or tissuetargeted and the specific makeup of the CRISPR/Cas9/gRNA. For example,in the EBV example discussed above, since lymphocytes are known forbeing resistant to lipofection, nucleofection (a combination ofelectrical parameters generated by a device called Nucleofector, withcell-type specific reagents to transfer a substrate directly into thecell nucleus and the cytoplasm) was necessitated for DNA delivery intothe Raji cells. The Lonza pmax promoter drives Cas9 expression as itoffered strong expression within Raji cells. 24 hours afternucleofection, obvious EGFP signals were observed from a smallproportion of cells through fluorescent microscopy. The EGFP-positivecell population decreased dramatically, however, <10% transfectionefficiency 48 hours after nucleofection was measured (FIG. 1B). A CRISPRplasmid that included the EBV origin of replication sequence, oriPyielded a transfection efficiency >60% (FIG. 1B).

Aspects of the invention utilize the CRISPR/Cas9/gRNA complexes for thetargeted delivery. Common known pathways include transdermal,transmucal, nasal, ocular and pulmonary routes. Drug delivery systemsmay include liposomes, proliposomes, microspheres, gels, prodrugs,cyclodextrins, etc. Aspects of the invention utilize nanoparticlescomposed of biodegradable polymers to be transferred into an aerosol fortargeting of specific sites or cell populations in the lung, providingfor the release of the drug in a predetermined manner and degradationwithin an acceptable period of time. Controlled-release technology(CRT), such as transdermal and transmucosal controlled-release deliverysystems, nasal and buccal aerosol sprays, drug-impregnated lozenges,encapsulated cells, oral soft gels, iontophoretic devices to administerdrugs through skin, and a variety of programmable, implanteddrug-delivery devices are used in conjunction with the complexes of theinvention of accomplishing targeted and controlled delivery.

v. Cut Viral Nucleic Acid

Once inside the cell, the CRISPR/Cas9/gRNA complexes target the viralgenome. In an aspect of the invention, the complexes are targeted toviral genomes. In addition to latent infections this invention can alsobe used to control actively replicating viruses by targeting the viralgenome before it is packaged or after it is ejected. In someembodiments, methods and compositions of the invention use a nucleasesuch as Cas9 to target latent viral genomes, thereby reducing thechances of proliferation. The nuclease may form a complex with a gRNA(e.g., crRNA+tracrRNA or sgRNA). The complex cuts the viral nucleic acidin a targeted fashion to incapacitate the viral genome. As discussedabove, the Cas9 endonuclease causes a double strand break in the viralgenome. By targeted several locations along the viral genome and causingnot a single strand break, but a double strand break, the genome iseffectively cut a several locations along the genome. In a preferredembodiment, the double strand breaks are designed so that smalldeletions are caused, or small fragments are removed from the genome sothat even if natural repair mechanisms join the genome together, thegenome is render incapacitated.

After introduction into a cell, the CRISPR/Cas9/gRNA complexes act onthe viral genome, genes, transcripts, or other viral nucleic acid. Thedouble-strand DNA breaks generated by CRISPR are repaired with smalldeletions. These deletions will disrupt the protein coding and hencecreate knockout effects.

The nuclease, or a gene encoding the nuclease, may be delivered into aninfected cell by transfection. For example, the infected cell can betransfected with DNA that encodes Cas9 and gRNA (on a single piece orseparate pieces). The gRNAs are designed to localize the Cas9endonuclease at one or several locations along the viral genome. TheCas9 endonuclease causes double strand breaks in the genome, causingsmall fragments to be deleted from the viral genome. Even with repairmechanisms, the deletions render the viral genome incapacitated.

vi. Host Genome

It will be appreciated that method and compositions of the invention canbe used to target viral nucleic acid without interfering with hostgenetic material. Methods and compositions of the invention employ atargeting moiety such as a guide RNA that has a sequence that hybridizesto a target within the viral sequence. Methods and compositions of theinvention may further use a targeted nuclease such as the cas9 enzyme,or a vector encoding such a nuclease, which uses the gRNA to bindexclusively to the viral genome and make double stranded cuts, therebyremoving the viral sequence from the host.

Where the targeting moiety includes a guide RNA, the sequence for thegRNA, or the guide sequence, can be determined by examination of theviral sequence to find regions of about 20 nucleotides that are adjacentto a protospacer adjacent motif (PAM) and that do not also appear in thehost genome adjacent to the protospacer motif.

Preferably a guide sequence that satisfies certain similarity criteria(e.g., at least 60% identical with identity biased toward regions closerto the PAM) so that a gRNA/cas9 complex made according to the guidesequence will bind to and digest specified features or targets in theviral sequence without interfering with the host genome. Preferably, theguide RNA corresponds to a nucleotide string next to a protospaceradjacent motif (PAM) (e.g., NGG, where N is any nucleotide) in the viralsequence. Preferably, the host genome lacks any region that (1) matchesthe nucleotide string according to a predetermined similarity criteriaand (2) is also adjacent to the PAM. The predetermined similaritycriteria may include, for example, a requirement of at least 12 matchingnucleotides within 20 nucleotides 5′ to the PAM and may also include arequirement of at least 7 matching nucleotides within 10 nucleotides 5′to the PAM. An annotated viral genome (e.g., from GenBank) may be usedto identify features of the viral sequence and finding the nucleotidestring next to a protospacer adjacent motif (PAM) in the viral sequencewithin a selected feature (e.g., a viral replication origin, a terminalrepeat, a replication factor binding site, a promoter, a codingsequence, or a repetitive region) of the viral sequence. The viralsequence and the annotations may be obtained from a genome database.

Where multiple candidate gRNA targets are found in the viral genome,selection of the sequence to be the template for the guide RNA may favorthe candidate target closest to, or at the 5′ most end of, a targetedfeature as the guide sequence. The selection may preferentially favorsequences with neutral (e.g., 40% to 60%) GC content. Additionalbackground regarding the RNA-directed targeting by endonuclease isdiscussed in U.S. Pub. 2015/0050699; U.S. Pub. 20140356958; U.S. Pub.2014/0349400; U.S. Pub. 2014/0342457; U.S. Pub. 2014/0295556; and U.S.Pub. 2014/0273037, the contents of each of which are incorporated byreference for all purposes. Due to the existence of human genomesbackground in the infected cells, a set of steps are provided to ensurehigh efficiency against the viral genome and low off-target effect onthe human genome. Those steps may include (1) target selection withinviral genome, (2) avoiding PAM+target sequence in host genome, (3)methodologically selecting viral target that is conserved acrossstrains, (4) selecting target with appropriate GC content, (5) controlof nuclease expression in cells, (6) vector design, (7) validationassay, others and various combinations thereof. A targeting moiety (suchas a guide RNA) preferably binds to targets within certain categoriessuch as (i) latency related targets, (ii) infection and symptom relatedtargets, and (iii) structure related targets.

A first category of targets for gRNA includes latency-related targets.The viral genome requires certain features in order to maintain thelatency. These features include, but not limited to, mastertranscription regulators, latency-specific promoters, signaling proteinscommunicating with the host cells, etc. If the host cells are dividingduring latency, the viral genome requires a replication system tomaintain genome copy level. Viral replication origin, terminal repeats,and replication factors binding to the replication origin are greattargets. Once the functions of these features are disrupted, the virusesmay reactivate, which can be treated by conventional antiviraltherapies.

A second category of targets for gRNA includes infection-related andsymptom-related targets. Virus produces various molecules to facilitateinfection. Once gained entrance to the host cells, the virus may startlytic cycle, which can cause cell death and tissue damage (HBV). Incertain cases, such as HPV16, cell products (E6 and E7 proteins) cantransform the host cells and cause cancers. Disrupting the key genomesequences (promoters, coding sequences, etc) producing these moleculescan prevent further infection, and/or relieve symptoms, if not curingthe disease.

A third category of targets for gRNA includes structure-related targets.Viral genome may contain repetitive regions to support genomeintegration, replication, or other functions. Targeting repetitiveregions can break the viral genome into multiple pieces, whichphysically destroys the genome.

Where the nuclease is a cas protein, the targeting moiety is a guideRNA. Each cas protein requires a specific PAM next to the targetedsequence (not in the guide RNA). This is the same as for human genomeediting. The current understanding the guide RNA/nuclease complex bindsto PAM first, then searches for homology between guide RNA and targetgenome. Sternberg et al., 2014, DNA interrogation by the CRISPRRNA-guided endonuclease Cas9, Nature 507(7490):62-67. Once recognized,the DNA is digested 3-nt upstream of PAM. These results suggest thatoff-target digestion requires PAM in the host DNA, as well as highaffinity between guide RNA and host genome right before PAM.

It may be preferable to use a targeting moiety that targets portions ofthe viral genome that are highly conserved. Viral genomes are much morevariable than human genomes. In order to target different strains, theguide RNA will preferably target conserved regions. As PAM is importantto initial sequence recognition, it is also essential to have PAM in theconserved region.

In a preferred embodiment, methods of the invention are used to delivera nucleic acid to cells. The nucleic acid delivered to the cells mayinclude a gRNA having the determined guide sequence or the nucleic acidmay include a vector, such as a plasmid, that encodes an enzyme thatwill act against the target genetic material. Expression of that enzymeallows it to degrade or otherwise interfere with the target geneticmaterial. The enzyme may be a nuclease such as the Cas9 endonuclease andthe nucleic acid may also encode one or more gRNA having the determinedguide sequence.

The gRNA targets the nuclease to the target genetic material. Where thetarget genetic material includes the genome of a virus, gRNAscomplementary to parts of that genome can guide the degredation of thatgenome by the nuclease, thereby preventing any further replication oreven removing any intact viral genome from the cells entirely. By thesemeans, latent viral infections can be targeted for eradication.

The host cells may grow at different rate, based on the specific celltype. High nuclease expression is necessary for fast replicating cells,whereas low expression help avoiding off-target cutting in non-infectedcells. Control of nuclease expression can be achieved through severalaspects. If the nuclease is expressed from a vector, having the viralreplication origin in the vector can increase the vector copy numberdramatically, only in the infected cells. Each promoter has differentactivities in different tissues. Gene transcription can be tuned bychoosing different promoters. Transcript and protein stability can alsobe tuned by incorporating stabilizing or destabilizing (ubiquitintargeting sequence, etc) motif into the sequence.

Specific promoters may be used for the gRNA sequence, the nuclease(e.g., cas9), other elements, or combinations thereof. For example, insome embodiments, the gRNA is driven by a U6 promoter. A vector may bedesigned that includes a promoter for protein expression (e.g., using apromoter as described in the vector sold under the trademark PMAXCLONINGby Lonza Group Ltd (Basel, Switzerland). A vector may be a plasmid(e.g., created by synthesis instrument 255 and recombinant DNA labequipment). In certain embodiments, the plasmid includes a U6 promoterdriven gRNA or chimeric guide RNA (sgRNA) and a ubiquitouspromoter-driven cas9. Optionally, the vector may include a marker suchas EGFP fused after the cas9 protein to allow for later selection ofcas9+ cells. It is recognized that cas9 can use a gRNA (similar to theCRISPR RNA (crRNA) of the original bacterial system) with acomplementary trans-activating crRNA (tracrRNA) to target viralsequences complementary to the gRNA. It has also been shown that cas9can be programmed with a single RNA molecule, a chimera of the gRNA andtracrRNA. The single guide RNA (sgRNA) can be encoded in a plasmid andtranscription of the sgRNA can provide the programming of cas9 and thefunction of the tracrRNA. See Jinek, 2012, A programmabledual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science337:816-821 and especially FIG. 5A therein for background.

Using the above principles, methods and compositions of the inventionmay be used to target viral nucleic acid in an infected host withoutadversely influencing the host genome.

For additional background see Hsu, 2013, DNA targeting specificity ofRNA-guided Cas9 nucleases, Nature Biotechnology 31(9):827-832; andJinek, 2012, A programmable dual-RNA-guided DNA endonuclease in adaptivebacterial immunity, Science 337:816-821, the contents of each of whichare incorporated by reference. Since the targeted locations are selectedto be within certain categories such as (i) latency related targets,(ii) infection and symptom related targets, or (iii) structure relatedtargets, cleavage of those sequences inactivates the virus and removesit from the host. Since the targeting RNA (the gRNA or sgRNA) isdesigned to satisfy according to similarity criteria that matches thetarget in the viral genetic sequence without any off-target matching thehost genome, the latent viral genetic material is removed from the hostwithout any interference with the host genome.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Targeting EBV

Burkitt's lymphoma cell lines Raji, Namalwa, and DG-75 were obtainedfrom ATCC and cultured in RPMI 1640 supplemented with 10% FBS and PSA,following ATCC recommendation. Human primary lung fibroblast IMR-90 wasobtained from Coriell and cultured in Advanced DMEM/F-12 supplementedwith 10% FBS and PSA.

Plasmids consisting of a U6 promoter driven chimeric guide RNA (sgRNA)and a ubiquitous promoter driven Cas9 were obtained from addgene, asdescribed by Cong L et al. (2013) Multiplex Genome Engineering UsingCRISPR/Cas Systems. Science 339:819-823. An EGFP marker fused after theCas9 protein allowed selection of Cas9-positive cells (FIG. 1A). Weadapted a modified chimeric guide RNA design for more efficient Pol-IIItranscription and more stable stem-loop structure (Chen B et al. (2013)Dynamic Imaging of Genomic Loci in Living Human Cells by an OptimizedCRISPR/Cas System. Cell 155:1479-1491).

We obtained pX458 from Addgene, Inc. A modified CMV promoter with asynthetic intron (pmax) was PCR amplified from Lonza control plasmidpmax-GFP. A modified guide RNA sgRNA(F+E) was ordered from IDT. EBVreplication origin oriP was PCR amplified from B95-8 transformedlymphoblastoid cell line GM12891. We used standard cloning protocols toclone pmax, sgRNA(F+E) and oriP to pX458, to replace the original CAGpromoter, sgRNA and f1 origin. We designed EBV sgRNA based on the B95-8reference, and ordered DNA oligos from IDT. The original sgRNA placeholder in pX458 serves as the negative control.

Lymphocytes are known for being resistant to lipofection, and thereforewe used nucleofection for DNA delivery into Raji cells. We chose theLonza pmax promoter to drive Cas9 expression as it offered strongexpression within Raji cells. We used the Lonza Nucleofector II for DNAdelivery. 5 million Raji or DG-75 cells were transfected with 5 ugplasmids in each 100-ul reaction. Cell line Kit V and program M-013 wereused following Lonza recommendation. For IMR-90, 1 million cells weretransfected with 5 ug plasmids in 100 ul Solution V, with program T-030or X-005. 24 hours after nucleofection, we observed obvious EGFP signalsfrom a small proportion of cells through fluorescent microscopy. TheEGFP-positive cell population decreased dramatically after that,however, and we measured <10% transfection efficiency 48 hours afternucleofection (FIG. 1 B). We attributed this transfection efficiencydecrease to the plasmid dilution with cell division. To activelymaintain the plasmid level within the host cells, we redesigned theCRISPR plasmid to include the EBV origin of replication sequence, oriP.With active plasmid replication inside the cells, the transfectionefficiency rose to >60% (FIG. 1B).

To design guide RNA targeting the EBV genome, we relied on the EBVreference genome from strain B95-8. We targeted six regions with sevenguide RNA designs for different genome editing purposes (FIG. 1C andTable S1).

TABLE S1 Guide RNA target sequences sgEBV1 (SEQ ID NO: 1)GCCCTGGACCAACCCGGCCC sgEBV2 (SEQ ID NO: 2) GGCCGCTGCCCCGCTCCGGG sgEBB3(SEQ ID NO: 3) GGAAGACAATGTGCCGCCA sgEBV4 (SEQ ID NO: 4)TCTGGACCAGAAGGCTCCGG sgEBV5 (SEQ ID NO: 5) GCTGCCGCGGAGGGTGATGA sgEBV6(SEQ ID NO: 6) GGTGGCCCACCGGGTCCGCT sgEBV7 (SEQ ID NO: 7)GTCCTCGAGGGGGCCGTCGC

EBNA1 is crucial for many EBV functions including gene regulation andlatent genome replication. We targeted guide RNA sgEBV4 and sgEBV5 toboth ends of the EBNA1 coding region in order to excise this wholeregion of the genome. Guide RNAs sgEBV1, 2 and 6 fall in repeat regions,so that the success rate of at least one CRISPR cut is multiplied. These“structural” targets enable systematic digestion of the EBV genome intosmaller pieces. EBNA3C and LMP1 are essential for host celltransformation, and we designed guide RNAs sgEBV3 and sgEBV7 to targetthe 5′ exons of these two proteins respectively.

EBV Genome Editing. The double-strand DNA breaks generated by CRISPR arerepaired with small deletions. These deletions will disrupt the proteincoding and hence create knockout effects. SURVEYOR assays confirmedefficient editing of individual sites (FIG. 5). Beyond the independentsmall deletions induced by each guide RNA, large deletions betweentargeting sites can systematically destroy the EBV genome. Guide RNAsgEBV2 targets a region with twelve 125-bp repeat units (FIG. 2A). PCRamplicon of the whole repeat region gave a ˜1.8-kb band (FIG. 2B). After5 or 7 days of sgEBV2 transfection, we obtained ˜0.4-kb bands from thesame PCR amplification (FIG. 2B). The ˜1.4-kb deletion is the expectedproduct of repair ligation between cuts in the first and the last repeatunit (FIG. 2A).

DNA sequences flanking sgRNA targets were PCR amplified with Phusion DNApolymerase. SURVEYOR assays were performed following manufacturer'sinstruction. DNA amplicons with large deletions were TOPO cloned andsingle colonies were used for Sanger sequencing. EBV load was measuredwith Taqman digital PCR on Fluidigm BioMark. A Taqman assay targeting aconserved human locus was used for human DNA normalization. 1 ng ofsingle-cell whole-genome amplification products from Fluidigm Cl wereused for EBV quantitative PCR.

We further demonstrated that it is possible to delete regions betweenunique targets (FIG. 2C). Six days after sgEBV4-5 transfection, PCRamplification of the whole flanking region (with primers EBV4F and 5R)returned a shorter amplicon, together with a much fainter band of theexpected 2 kb (FIG. 2D). Sanger sequencing of amplicon clones confirmedthe direct connection of the two expected cutting sites (FIG. 2F). Asimilar experiment with sgEBV3-5 also returned an even larger deletion,from EBNA3C to EBNA1 (FIG. 2D-E).

Additional information such as primer design is shown in Wang and Quake,2014, RNA-guided endonuclease provides a therapeutic strategy to curelatent herpesviridae infection, PNAS 111(36):13157-13162 and in theSupporting Information to that article published online at the PNASwebsite, and the contents of both of those documents are incorporated byreference for all purposes.

Cell Proliferation Arrest With EBV Genome Destruction. Two days afterCRISPR transfection, we flow sorted EGFP-positive cells for furtherculture and counted the live cells daily. As expected, cells treatedwith Cas9 plasmids which lacked oriP or sgEBV lost EGFP expressionwithin a few days and proliferated with a rate similar rate to theuntreated control group (FIG. 3A). Plasmids with Cas9-oriP and ascrambled guide RNA maintained EGFP expression after 8 days, but did notreduce the cell proliferation rate. Treatment with the mixed cocktailsgEBV 1-7 resulted in no measurable cell proliferation and the totalcell count either remained constant or decreased (FIG. 3A). Flowcytometry scattering signals clearly revealed alterations in the cellmorphology after sgEBV 1-7 treatment, as the majority of the cellsshrank in size with increasing granulation (FIG. 3B-D, population P4 toP3 shift). Cells in population P3 also demonstrated compromised membranepermeability by DAPI staining (FIG. 3E-G). To rule out the possibilityof CRISPR cytotoxicity, especially with multiple guide RNAs, weperformed the same treatment on two other samples: the EBV-negativeBurkitt's lymphoma cell line DG-75 (FIG. 6) and primary human lungfibroblast IMR90 (FIG. 7). Eight and nine days after transfection thecell proliferation rates did not change from the untreated controlgroups, suggesting neglectable cytotoxicity.

Previous studies have attributed the EBV tumorigenic ability to itsinterruption of host cell apoptosis (Ruf I K et al. (1999) Epstein-BarrVirus Regulates c-MYC, Apoptosis, and Tumorigenicity in BurkittLymphoma. Molecular and Cellular Biology 19:1651-1660). Suppressing EBVactivities may therefore restore the apoptosis process, which couldexplain the cell death observed in our experiment Annexin V stainingrevealed a distinct subpopulation of cells with intact cell membrane butexposed phosphatidylserine, suggesting cell death through apoptosis(FIG. 3E-G). Bright field microscopy showed obvious apoptotic cellmorphology (FIG. 3H-I) and fluorescent staining demonstrated drastic DNAfragmentation (FIG. 3J-M). Altogether this evidence suggests restorationof the normal host cell apoptosis pathway after EBV genome destruction.

Complete Clearance Of EBV In A Subpopulation. To study the potentialconnection between cell proliferation arrest and EBV genome editing, wequantified the EBV load in different samples with digital PCR targetingEBNA1. Another Taqman assay targeting a conserved human somatic locusserved as the internal control for human DNA normalization. On average,each untreated Raji cell has 42 copies of EBV genome (FIG. 4A). Cellstreated with a Cas9 plasmid that lacked oriP or sgEBV did not have anobvious difference in EBV load difference from the untreated control.Cells treated with a Cas9-plasmid with oriP but no sgEBV had an EBV loadthat was reduced by ˜50%. In conjunction with the prior observation thatcells from this experiment did not show any difference in proliferationrate, we interpret this as likely due to competition for EBNA1 bindingduring plasmid replication. The addition of the guide RNA cocktailsgEBV1-7 to the transfection dramatically reduced the EBV load. Both thelive and dead cells have >60% EBV decrease comparing to the untreatedcontrol.

Although we provided seven guide RNAs at the same molar ratio, theplasmid transfection and replication process is likely quite stochastic.Some cells will inevitably receive different subsets or mixtures of theguide RNA cocktail, which might affect the treatment efficiency. Tocontrol for such effects, we measured EBV load at the single cell levelby employing single-cell whole-genome amplification with an automatedmicrofluidic system. We loaded freshly cultured Raji cells onto themicrofluidic chip and captured 81 single cells (FIG. 4B). For thesgEBV1-7 treated cells, we flow sorted the live cells eight days aftertransfection and captured 91 single cells (FIG. 4C). Followingmanufacturer's instruction, we obtained ˜150 ng amplified DNA from eachsingle cell reaction chamber. For quality control purposes we performed4-loci human somatic DNA quantitative PCR on each single cellamplification product (Wang J, Fan H C, Behr B, Quake S R (2012)Genome-wide single-cell analysis of recombination activity and de novomutation rates in human sperm. Cell 150:402-412) and required positiveamplification from at least one locus. 69 untreated single-cell productspassed the quality control and displayed a log-normal distribution ofEBV load (FIG. 4D) with almost every cell displaying significant amountsof EBV genomic DNA. We calibrated the quantitative PCR assay with asubclone of Namalwa Burkitt's lymphoma cells, which contain a singleintegrated EBV genome. The single-copy EBV measurements gave a Ct of29.8, which enabled us to determine that the mean Ct of the 69 Rajisingle cell samples corresponded to 42 EBV copies per cells, inconcordance with the bulk digital PCR measurement. For the sgEBV1-7treated sample, 71 single-cell products passed the quality control andthe EBV load distribution was dramatically wider (FIG. 4E). While 22cells had the same EBV load as the untreated cells, 19 cells had nodetectable EBV and the remaining 30 cells displayed dramatic EBV loaddecrease from the untreated sample.

Essential Targets For EBV Treatment. The seven guide RNAs in our CRISPRcocktail target three different categories of sequences which areimportant for EBV genome structure, host cell transformation, andinfection latency, respectively. To understand the most essentialtargets for effective EBV treatment, we transfected Raji cells withsubsets of guide RNAs. Although sgEBV4/5 reduced the EBV genome by 85%,they could not suppress cell proliferation as effectively as the fullcocktail (FIG. 3A). Guide RNAs targeting the structural sequences(sgEBV1/2/6) could stop cell proliferation completely, despite noteliminating the full EBV load (26% decrease). Given the high efficiencyof genome editing and the proliferation arrest (FIG. 2), we suspect thatthe residual EBV genome signature in sgEBV1/2/6 was not due to intactgenomes but to free-floating DNA that has been digested out of the EBVgenome, i.e. as a false positive. We conclude that systematicdestruction of EBV genome structure appears to be more effective thantargeting specific key proteins for EBV treatment.

Example 2 Targeting Hepatitis B Virus (HBV)

Methods and materials of the present invention may be used to applytargeted endonuclease to specific genetic material such as a latentviral genome like the hepatitis B virus (HBV). The invention furtherprovides for the efficient and safe delivery of nucleic acid (such as aDNA plasmid) into target cells (e.g., hepatocytes). In one embodiment,methods of the invention use hydrodynamic gene delivery to target HBV.

FIG. 10 diagrams the HBV genome. It may be preferable to receiveannotations for the HBV genome (i.e., that identify important featuresof the genome) and choose a candidate for targeting by enzymaticdegredation that lies within one of those features, such as a viralreplication origin, a terminal repeat, a replication factor bindingsite, a promoter, a coding sequence, and a repetitive region.

HBV, which is the prototype member of the family Hepadnaviridae, is a 42nm partially double stranded DNA virus, composed of a 27 nm nucleocapsidcore (HBcAg), surrounded by an outer lipoprotein coat (also calledenvelope) containing the surface antigen (HBsAg). The virus includes anenveloped virion containing 3 to 3.3 kb of relaxed circular, partiallyduplex DNA and virion-associated DNA-dependent polymerases that canrepair the gap in the virion DNA template and has reverse transcriptaseactivities. HBV is a circular, partially double-stranded DNA virus ofapproximately 3200 bp with four overlapping ORFs encoding the polymerase(P), core (C), surface (S) and X proteins. In infection, viralnucleocapsids enter the cell and reach the nucleus, where the viralgenome is delivered. In the nucleus, second-strand DNA synthesis iscompleted and the gaps in both strands are repaired to yield acovalently closed circular DNA molecule that serves as a template fortranscription of four viral RNAs that are 3.5, 2.4, 2.1, and 0.7 kblong. These transcripts are polyadenylated and transported to thecytoplasm, where they are translated into the viral nucleocapsid andprecore antigen (C, pre-C), polymerase (P), envelope L (large), M(medium), S (small)), and transcriptional transactivating proteins (X).The envelope proteins insert themselves as integral membrane proteinsinto the lipid membrane of the endoplasmic reticulum (ER). The 3.5 kbspecies, spanning the entire genome and termed pregenomic RNA (pgRNA),is packaged together with HBV polymerase and a protein kinase into coreparticles where it serves as a template for reverse transcription ofnegative-strand DNA. The RNA to DNA conversion takes place inside theparticles.

Numbering of basepairs on the HBV genome is based on the cleavage sitefor the restriction enzyme EcoR1 or at homologous sites, if the EcoR1site is absent. However, other methods of numbering are also used, basedon the start codon of the core protein or on the first base of the RNApregenome. Every base pair in the HBV genome is involved in encoding atleast one of the HBV protein. However, the genome also contains geneticelements which regulate levels of transcription, determine the site ofpolyadenylation, and even mark a specific transcript for encapsidationinto the nucleocapsid. The four ORFs lead to the transcription andtranslation of seven different HBV proteins through use of varyingin-frame start codons. For example, the small hepatitis B surfaceprotein is generated when a ribosome begins translation at the ATG atposition 155 of the adw genome. The middle hepatitis B surface proteinis generated when a ribosome begins at an upstream ATG at position 3211,resulting in the addition of 55 amino acids onto the 5′ end of theprotein.

ORF P occupies the majority of the genome and encodes for the hepatitisB polymerase protein. ORF S encodes the three surface proteins. ORF Cencodes both the hepatitis e and core protein. ORF X encodes thehepatitis B X protein. The HBV genome contains many important promoterand signal regions necessary for viral replication to occur. The fourORFs transcription are controlled by four promoter elements (preS1,preS2, core and X), and two enhancer elements (Enh I and Enh II). AllHBV transcripts share a common adenylation signal located in the regionspanning 1916-1921 in the genome. Resulting transcripts range from 3.5nucleotides to 0.9 nucleotides in length. Due to the location of thecore/pregenomic promoter, the polyadenylation site is differentiallyutilized. The polyadenylation site is a hexanucleotide sequence (TATAAA)as opposed to the canonical eukaryotic polyadenylation signal sequence(AATAAA). The TATAAA is known to work inefficiently (9), suitable fordifferential use by HBV.

There are four known genes encoded by the genome, called C, X, P, and S.The core protein is coded for by gene C (HBcAg), and its start codon ispreceded by an upstream in-frame AUG start codon from which the pre-coreprotein is produced. HBeAg is produced by proteolytic processing of thepre-core protein. The DNA polymerase is encoded by gene P. Gene S is thegene that codes for the surface antigen (HBsAg). The HBsAg gene is onelong open reading frame but contains three in-frame start (ATG) codonsthat divide the gene into three sections, pre-S1, pre-S2, and S. Becauseof the multiple start codons, polypeptides of three different sizescalled large, middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S) areproduced. The function of the protein coded for by gene X is not fullyunderstood but it is associated with the development of liver cancer. Itstimulates genes that promote cell growth and inactivates growthregulating molecules.

With reference to FIG. 10, HBV starts its infection cycle by binding tothe host cells with PreS1. Guide RNA against PreS1 locates at the 5′ endof the coding sequence. Endonuclease digestion will introduceinsertion/deletion, which leads to frame shift of PreS 1 translation.HBV replicates its genome through the form of long RNA, with identicalrepeats DR1 and DR2 at both ends, and RNA encapsidation signal epsilonat the 5′ end. The reverse transcriptase domain (RT) of the polymerasegene converts the RNA into DNA. Hbx protein is a key regulator of viralreplication, as well as host cell functions. Digestion guided by RNAagainst RT will introduce insertion/deletion, which leads to frame shiftof RT translation. Guide RNAs sgHbx and sgCore can not only lead toframe shift in the coding of Hbx and HBV core protein, but also deletionthe whole region containing DR2-DR1-Epsilon. The four sgRNA incombination can also lead to systemic destruction of HBV genome intosmall pieces.

HBV replicates its genome by reverse transcription of an RNAintermediate. The RNA templates is first converted into single-strandedDNA species (minus-strand DNA), which is subsequently used as templatesfor plus-strand DNA synthesis. DNA synthesis in HBV use RNA primers forplus-strand DNA synthesis, which predominantly initiate at internallocations on the single-stranded DNA. The primer is generated via anRNase H cleavage that is a sequence independent measurement from the 5′end of the RNA template. This 18 nt RNA primer is annealed to the 3′ endof the minus-strand DNA with the 3′ end of the primer located within the12 nt direct repeat, DR1. The majority of plus-strand DNA synthesisinitiates from the 12 nt direct repeat, DR2, located near the other endof the minus-strand DNA as a result of primer translocation. The site ofplus-strand priming has consequences. In situ priming results in aduplex linear (DL) DNA genome, whereas priming from DR2 can lead to thesynthesis of a relaxed circular (RC) DNA genome following completion ofa second template switch termed circularization. It remains unclear whyhepadnaviruses have this added complexity for priming plus-strand DNAsynthesis, but the mechanism of primer translocation is a potentialtherapeutic target. As viral replication is necessary for maintenance ofthe hepadnavirus (including the human pathogen, hepatitis B virus)chronic carrier state, understanding replication and uncoveringtherapeutic targets is critical for limiting disease in carriers.

In some embodiments, systems and methods of the invention target the HBVgenome by finding a nucleotide string within a feature such as PreS1.

Guide RNA against PreS1 locates at the 5′ end of the coding sequence.Thus it is a good candidate for targeting because it represents one ofthe 5′-most targets in the coding sequence. Endonuclease digestion willintroduce insertion/deletion, which leads to frame shift of PreS1translation. HBV replicates its genome through the form of long RNA,with identical repeats DR1 and DR2 at both ends, and RNA encapsidationsignal epsilon at the 5′ end.

The reverse transcriptase domain (RT) of the polymerase gene convertsthe RNA into DNA. Hbx protein is a key regulator of viral replication,as well as host cell functions. Digestion guided by RNA against RT willintroduce insertion/deletion, which leads to frame shift of RTtranslation.

Guide RNAs sgHbx and sgCore can not only lead to frame shift in thecoding of Hbx and HBV core protein, but also deletion the whole regioncontaining DR2-DR1-Epsilon. The four sgRNA in combination can also leadto systemic destruction of HBV genome into small pieces. In someembodiments, method of the invention include creating one or severalguide RNAs against key features within a genome such as the HBV genomeshown in FIG. 10.

FIG. 10 shows key parts in the HBV genome targeted by CRISPR guide RNAs.To achieve the CRISPR activity in cells, expression plasmids coding cas9and guide RNAs are delivered to cells of interest (e.g., cells carryingHBV DNA). To demonstrate in an in vitro assay, anti-HBV effect may beevaluated by monitoring cell proliferation, growth, and morphology aswell as analyzing DNA integrity and HBV DNA load in the cells.

The described method may be validated using an in vitro assay. Todemonstrate, an in vitro assay is performed with cas9 protein and DNAamplicons flanking the target regions. Here, the target is amplified andthe amplicons are incubated with cas9 and a gRNA having the selectednucleotide sequence for targeting. As shown in FIG. 11, DNAelectrophoresis shows strong digestion at the target sites.

FIG. 11 shows a gel resulting from an in vitro CRISPR assay against HBV.Lanes 1, 3, and 6: PCR amplicons of HBV genome flanking RT, Hbx-Core,and PreS1. Lane 2, 4, 5, and 7: PCR amplicons treated with sgHBV-RT,sgHBV-Hbx, sgHBV-Core, sgHBV-PreS1. The presence of multiple fragmentsespecially visible in lanes 5 and 7 show that sgHBV-Core and sgHBV-PreS1 provide especially attractive targets in the context of HBV and thatuse of systems and methods of the invention may be shown to be effectiveby an in vitro validation assay.

What is claimed is:
 1. A method for treating a viral infection, themethod comprising: introducing into a cell a nuclease and asequence-specific targeting moiety; targeting the nuclease to a viralnucleic acid by means of the sequence-specific targeting moiety; andcleaving the viral nucleic acid with the nuclease without interferingwith the host genome.
 2. The method of claim 1, wherein the nuclease isselected from the group consisting of a zinc-finger nuclease, atranscription activator-like effector nuclease, and a meganuclease. 3.The method of claim 1, wherein the nuclease is a Cas9 nuclease and thesequence-specific targeting moiety comprises a guide RNA.
 4. The methodof claim 1, wherein the viral nucleic acid is latent in a host cell. 5.The method of claim 1, wherein said cleaving step comprises creating adouble-strand break in said viral nucleic acid.
 6. The method of claim1, further comprising the step of inserting a polynucleotide into theviral nucleic acid.
 7. The method of claim 1, wherein the viral nucleicacid is from a virus selected from the group consisting of adenovirus,herpes simplex virus, varicella-zoster virus, Epstein-barr virus, humancytomegalovirus, human herpesvirus type 8, human papillomavirus, BKvirus JC virus, smallpox, hepatitis B virus, human bocavirus,parvovirus, B19, human astrovirus, Norwalk virus, coxsackievirus,hepatitis A virus, poliovirus, rhinovirus, sever acute respiratorysyndrome virus, hepatitis C virus, yellow fever virus, dengue virus,west nile virus, rubella virus, hepatitis E virus, humanimmunodeficiency virus, influenza virus, guanarito virus, junin virus,lassa virus, machupo virus, sabia virus, Crimean-congo hemorrhagic fevervirus, ebola virus, Marburg virus, measles virus, mumps virus,parainfluenza virus, respiratory syncytial virus, human metapnemovirus,Hendra virus, nipah virus, rabies virus, hepatitis D virus, rotavirus,orbivirus, coltivirus, and banna virus.
 8. The method of claim 1,wherein said introducing step comprises introducing into the cell avector that encodes the nuclease and the sequence-specific targetingmoiety.
 9. The method of claim 8, wherein the vector is a viral vectorselected from the group consisting of retrovirus, lentivirus,adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia virus, andadeno-associated viruses.
 10. The method of claim 1, wherein saidintroducing step comprises introducing into the cell a non-viral vector.11. The method of claim 10, wherein said non-viral vector is selectedfrom the group consisting of a nanoparticle, a cationic lipid, acationic polymer, a metallic nanoparticle, a nanorod, a liposome,microbubbles, a cell-penetrating peptide, and a liposphere.
 12. Themethod of claim 10, wherein the non-viral vector comprisespolyethyleneglycol.